TGx-DDI (toxicogenomic DNA damage-inducing) biomarker validation: multi-site ring trial supporting regulatory use

TL;DR

The TGx-DDI biomarker improves genotoxicity testing by accurately identifying DNA-damaging compounds using gene expression data from human cells.

Contribution

TGx-DDI is a validated transcriptomic biomarker with high reproducibility and specificity for genotoxicity assessment across multiple laboratories.

Findings

TGx-DDI achieved 100% sensitivity, 86% specificity, and 91% accuracy in classifying DNA damage-inducing compounds.

High interlaboratory agreement and strong transcriptomic data correlation were observed across four sites.

The biomarker reliably classified compounds regardless of the study site, demonstrating robustness and reproducibility.

Abstract

Standard in vitro genotoxicity assays often suffer from low specificity, leading to irrelevant positive findings that require costly in vivo follow-up studies. The TGx-DDI (Toxicogenomic DNA Damage-Inducing) transcriptomic biomarker was developed to address this limitation by identifying DNA damage-inducing compounds through gene expression profiling in human TK6 lymphoblastoid cells. To qualify TGx-DDI as a reliable, reproducible biomarker for augmenting genotoxicity hazard assessment, a multi-site ring-trial was conducted across four laboratories using 14 blinded test compounds and standardized protocols. TK6 cells were exposed to three concentrations of each compound, followed by RNA extraction and digital nucleic acid counting using the NanoString nCounter platform. A three-pronged bioinformatics approach—Nearest Shrunken Centroid Probability Analysis, Principal Component Analysis, and Hierarchical Clustering—was used to assign DDI or non-DDI classifications. TGx-DDI demonstrated 100% sensitivity, 86% specificity, and 91% accuracy in distinguishing DDI from non-DDI compounds under validated test conditions. High interlaboratory concordance was observed (agreement coefficients ≥0.61), and transcriptomic data showed strong cross-site correlation (Pearson r > 0.84). The biomarker reproducibly classified test agents even when conducted across study sites. These results demonstrate that TGx-DDI is a robust and reproducible transcriptomic biomarker that enhances the specificity of genotoxicity testing by distinguishing biologically relevant DNA damage responses. Its integration into genotoxicity testing strategies can support regulatory decision-making, reduce unnecessary animal use, and improve the assessment of human health risks.

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